Three distinct dissemination-related phenotypes have been identified in cells of the mouse B16 melanoma: tumorigenicity, ability to spontaneously metastasize to lungs and other organs, and ability to form colonies in organs following intravenous injection of cells. From a null (tumorigenic but nonmetastatic and noncolonizing) "progenitor" cell (clone G3), a spontaneous metastasis model system was derived, consisting of metastatic subclones generated by phenotypic diversification in monolayer culture (G3.5) and within a subcutaneous tumor transplant (G3.12), a null subclone produced by long-term culturing (G3.15), and a lung-colonizing subclone generated within a tumor transplant (G3.26). Despite these innate phenotypes, all clones could express metastatic activity, at least transiently, under various experimental conditions that resulted in alteration of tumor growth rate; an intermediate rate seems to correlate with metastatic behavior. This model system, and especially clones G3.12 and G3.26, will be used in a comparative pathobiological investigation of the hypothesis that the intratumor environment created by certain tumor growth rates is critical for metastasis to occur, and that metastasis results from passive dissemination of quiescent and/or hypoxic cells through partially disrupted blood vessels and lymphatic channels within necrotic or quasi-necrotic tumor regions. Within viable tumor cell populations purified from subcutaneous tumors, cell cycle kinetics will be examined by flow cytometry, and the proportion of viable cells represented by quiescent cells will be determined. The proportion of radioresistant and hypoxic tumor cells will be determined by assessment of relative tumor cell radiosensitivity in intact tumors and by incorporation of a radiolabeled hypoxic cell radiosensitizer, 14C-labeled misonidazole. Localization of hypoxic cells within tumors will be achieved by autoradiography, and necrotic tumor regions, including affected blood vessels, will be quantitated morphometrically. The integrity of intratumor blood vessels will be determined by morphometric analysis of tumors perfused with india ink tracer and by examination of vascular casts of tumors. During initial metastatic dissemination to the lungs, the approximate numbers of viable tumor cells entering the lungs, and the relative timing of that dissemination, will be monitored by excising and culturing the lungs at various intervals during subcutaneous tumor progression.